Characterization of protoporphyrin-IX-positive EVs following 5-ALA use in patients with malignant glioma
Pamela Jones, Anudeep Yekula, Bob Carter, Leonora Balaj
Introduction: Malignant gliomas are rapidly progressive brain tumors with high mortality. Fluorescence guided surgery (FGS) with 5-aminolevulinic acid (5-ALA) provides fluorescent delineation of malignant tissue, which helps achieve maximum safe resection. 5-ALA-based fluorescence is due to preferential accumulation of the fluorophore protoporphyrin-IX (PpIX) in malignant glioma tissue. Additionally, gliomas cells release extracellular vesicles (EVs) which carry biomarkers of disease.
Objective: Our objectives were to first, determine if PpIX-positive EVs can be found from 5-ALA dosed Gli36 cells, determine if PpIX-positive EV signal can be detected up in plasma of glioma-bearing mice and not in control mice, and finally, to capture and enumerate PpIX-positive EVs from human plasma and correlate them as biomarkers of malignant glioma.
Methods: We used imaging flow cytometry to characterize PpIX-positive EVs released from 5-ALA-dosed glioma cells, 5-ALA-dosed glioma-bearing xenograft models and control mice, as well as patients with malignant glioma undergoing 5-ALA FGS.
Results: We show that 5-ALA-dosed glioma cells produce PpIX-positive EVs as compared 5-ALA-dosed human brain microvascular endothelial cells. We also found PpIX-positive EVs in the plasma from 5-ALA-dosed glioma-bearing mice compared to no rise in signal from the 5-ALA-dosed control mice. Finally, we show that plasma of patients with avidly fluorescent tumors under FGS contain circulating PpIX-positive EVs at levels significantly higher than their pre-dosing background and this rise in signal is positively correlated with enhancing tumor volumes (Figure 1).
Conclusions: Our findings highlight the potential of plasma-derived PpIX-positive EV-based diagnostics for malignant gliomas, offering a novel liquid biopsy platform for confirming and monitoring tumor status.